The overall goal of this application is to elucidate different mechanisms for negative regulation of Notch signaling. Notch is the eponymous receptor in a major signaling system for cell-cell interactions and cell fate specification in animl development. Regulating Notch signaling appropriately--in space, time, strength or duration--is critically important for normal development. Furthermore, aberrant Notch activity has been implicated in many different cancers, as well as in developmental, immune, and neurological disorders. Thus, the proposed work has many implications for human health. The deeper understanding of developmental mechanism has great potential for developing diagnostic and therapeutic tools for human disease, a central mission of the NIH. Our past work on LIN-12/Notch signaling in C. elegans has afforded many fundamental insights into conserved roles, mechanisms, and regulation of Notch signaling. We expect that our continued work in this system will continue to reveal mechanisms of general relevance, and particular aspects of experimental design were chosen to maximize this prospect. Each aim of this application explores different ways LIN-12/Notch activity and stability is modulated in a textbook developmental paradigm in which six multipotential precursor cells adopt one of three distinct fates through LIN-12/Notch-mediated cell-cell interactions. The patterning is precise and robust, reflecting tight spatial and temporal control of LIN-12/Notch activity and multiple modes for integrating LIN-12/Notch activity with other conserved signaling pathways. In Aim 1, we propose to characterize new kinases we identified in a targeted screen for negative regulators and another gene, first identified by a mutation in a cancer patient, we showed acts as a negative regulator in C. elegans. To assess their functional conservation in Notch regulation, we will perform a human cell assay. In Aim 2, we propose to investigate LIN-12/Notch endocytic trafficking and its negative regulation by EGF Receptor activity in developmental patterning. EGF Receptor, like Notch, is an important oncogene, and crosstalk between these pathways occurs in cancer as well as in normal development. In Aim 3, we propose to elucidate how activated nuclear LIN-12/Notch is inhibited by EGF receptor during developmental patterning and by Insulin/Insulin-like Signaling during quiescence in response to unfavorable environment. Quiescence is a fundamental property of critical importance for human health, allowing stem cells to persist over prolonged periods in a competent state so as to be available to repopulate tissues when cells are lost to aging, injury or disease, and for cancer, quiescent stem-like cells are believed to be resistant to standard chemotherapy. Together, these aims will provide much new information about negative regulation of a fundamental signaling pathway in development and disease, in accord with the mission of the NIH.